Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV
disinfection
Michael R. Templeton*, Robert C. Andrews, and Ron Hofmann
Department of Civil Engineering, University of Toronto35 St. George Street, Toronto, Ontario, Canada M5S 1A4
*Corresponding Author Current Address: Tel. +44-207-594-6099, Fax. +44-207-594-6124, Department of Civil and Environmental Engineering, Imperial College London, South Kensington campus, London, United Kingdom SW7 2AZ Email address: [email protected].
Submitted for publication in Water Research
Submitted 17 October 2005
Re-submitted with corrections January 2006
Re-submitted again with corrections February 2007
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Abstract
This bench-scale study investigated the passage of particle-associated bacteriophage through a
dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent
ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered.
The water matrix was de-chlorinated tap water with either kaolin or Aldrich® humic acid (AHA)
added and coagulated with alum to form floc before filtration. The turbidity of the influent
flocculated water was 6.4 ± 1.5 NTU. Influent and filter effluent turbidity and particle counts
were measured as well as headloss across the filter media. Filter effluent samples were collected
for phage enumeration during three filter cycle stages: (i) filter ripening, (ii) stable operation, and
(iii) end of filter cycle. Stable filter operation was defined according to a filter effluent turbidity
goal of < 0.3 NTU. Influent and filter effluent samples were subsequently exposed to UV light
(254 nm) at 40 mJ/cm2 using a low pressure UV collimated beam. The study found statistically
significant differences (α = 0.05) in the quantity of particle-associated phage present in the filter
effluent during the three stages of filtration.. There was reduced UV disinfection efficiency due
to the presence of particle-associated phage in the filter effluent in trials with bacteriophage MS2
and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation
of phage relative to particle-free control conditions for both phages. Trends in filter effluent
turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The
results therefore suggest that maintenance of optimum filtration conditions upstream of UV
disinfection is a critical barrier to particle-associated viruses.
Keywords: Filtration; ultraviolet (UV) disinfection; virus; bacteriophage; particle-association.
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1. Introduction
Viruses are frequently associated with particles in natural waters and wastewaters due to
electrostatic attraction and hydrophobic interactions (Gerba, 1984). Potential adsorbents of
viruses include sand, pure clays (e.g. montmorillonite, kaolinite, bentonite), bacterial cells,
naturally occurring suspended colloids, sludge particles, and estuarine silts and sediments
(Bitton, 1975; Moore et al., 1975; Sakoda et al., 1997; Meschke and Sobsey, 1998). In addition,
viruses may be discharged into natural waters already associated with solids such as fecal matter
(Hejkal et al., 1979; Gerba, 1984). Viral particle-association has been shown to enhance virus
survival in the environment and may provide protection from chemical disinfection (Stagg et al.,
1977; Hejkal et al., 1979; Babich and Stotzky, 1980). The physical removal of particles that may
harbor viruses from disinfectants is therefore thought to be a crucial barrier against the viral
contamination of drinking water, especially when treating source waters of variable particulate
quality. Further, virus reduction is an important consideration in water reuse, so it is critical to
gain an understanding of any factors that may limit virus reduction (e.g. particle-association) or
specific events during treatment that may pose a challenge to virus reduction.
A number of studies have investigated the removal of pathogens by granular media filtration
and many have focused on the removal of the chlorine-resistant protozoan parasites
Cryptosporidium and Giardia (Al-Ani et al., 1986; Huck et al., 2002; Harrington et al., 2003). It
has also been shown that optimized granular media filtration preceded by
coagulation/flocculation can effectively remove viruses (Rao et al., 1988; Nasser et al., 1995)
and viral indicators such as bacteriophage MS2 (Huck et al., 2001; Harrington et al., 2003;
Xagoraraki et al., 2004). However, the removal of viruses that are associated with very fine
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particles (< 1-2 µm) that are potentially difficult to remove by filtration, especially at certain so-
called ‘off-spec’ stages of the filter trial (ripening, end of cycle), has not been investigated in
detail. The passage of these potentially “disinfectant-shielded” viruses through filters is of
greater concern than the breakthrough of dispersed (i.e. non-particle-associated) viruses which
are inactivated relatively easily by most primary disinfectants.
Filter effluent turbidity is a standard measure of filter performance prescribed by many
regulations in North America (e.g. USEPA 2006). Turbidity breakthrough and/or the
concentration of particles per unit volume of a certain size range (e.g. 2-10 µm) are typically
used to determine the end of the filtration cycle (Kawamura, 1999). Filtration rate, headloss, and
filter run time can also be used to define end of cycle (Castro et al., 2005). In addition, Huck et
al. (2001) developed a turbidity robustness index (TRI) in an attempt to quantify the stability and
quality of filter operation. This index incorporates the target turbidity goal (Tgoal), representing a
plant-specific performance goal for filter effluent turbidity (e.g. 0.1 NTU), the average turbidity
achieved by the filter in a cycle (T50), and the peak turbidity encountered during a cycle (T95). A
formula to calculate overall filter stability with respect to turbidity reduction is shown below:
A lower value of the index indicates a treatment process which is meeting the water quality
goal with relatively low variation (Huck et al., 2001). For example, if a treatment goal (e.g. 0.1
NTU) is always met and there is no deviation from that goal, the value of the robustness index
would be 1.0 (Huck et al., 2001). On the other hand, a higher robustness index value indicates
that the treatment is either not meeting its goal (on average) or the variability is high (Huck et al.,
2001). While this index yields information about the stability of filters in reducing turbidity, it
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was uncertain whether filters optimized for turbidity removal are simultaneously optimized for
removal of dispersed or particle-associated viruses. For example, studies have shown that
reduction of turbidity and particle counts are not necessarily indicative of the removal of
Cryptosporidium by treatment processes (LeChevallier et al., 1991; LeChevallier and Norton,
1992; Nieminski and Ongerth, 1995; Emelko, 2001).
Previous studies have also investigated when the breakthrough of pathogens reaches a peak
during a filter cycle (Emelko, 2001; Huck et al., 2001). Emelko (2001) reported that removal of
Cryptosporidium parvum was 0.5- to 1-log lower during filter ripening versus during stable
operation, and deteriorated by 3- to 4-log during end of cycle breakthrough conditions, even
though filter effluent turbidity remained below < 0.1 NTU. This is an example of the
shortcomings of filter effluent turbidity measurement as a direct indication of pathogen removal
by filters. The same may be true of the filter removal of particle-associated viruses.
Gerba (1984) divided a number of viruses into categories based on their empirically observed
adsorption to different types of particles. Bacteriophages MS2 and T4 were categorized as being
representative of the solid-adsorption behavior of the two largest categories of human viruses in
the study described in Gerba (1984). Therefore, by using both of these phages as viral surrogates,
it is anticipated that the adsorption behavior (i.e. to particles, to filter media) of a broad spectrum
of human viruses may be represented.
Synthetic water matrices are often used in coagulation and filtration studies such that
experimental variables (e.g. particle concentration, composition) can be controlled. For example,
kaolin clay (Bitton et al., 1972; Ohgaki and Mongkonsiri, 1990; Barbeau et al., 2004) and
Aldrich® humic acid powder (Bitton et al., 1972; Nasser et al., 1995) have been used in previous
studies to represent inorganic colloids and organic matter in surface waters, respectively.
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The present study was intended to complement an earlier, fundamental study by the authors
which investigated the particle characteristics that enhance virus survival during UV disinfection
(Templeton et al. 2005). That study found that UV-absorbing organic particles shield particle-
associated phage from UV light, while inorganic kaolin clay particles do not. The present study
adds to that information by considering the ability of granular media filtration, which often
precedes UV disinfection in surface water treatment, to remove particle-associated viruses, and
assess when in the filter cycle these viruses may pass through and potentially impact subsequent
UV disinfection. Further, this study considers turbidity levels that are more representative of
water treatment practice than those considered in the previous study, which focused on gaining a
fundamental understanding of the impact of specific particle characteristics on UV disinfection
and considered much higher turbidities (up to 100 NTU) than would typically be encountered.
The specific objectives of this study were:
(i) To determine to what extent particle-associated viral surrogates (bacteriophage MS2,
bacteriophage T4) pass through dual-media filters during stable operation;
(ii) To assess when in the filter cycle the breakthrough of particle-associated phage peaks;
(iii) To assess whether removal of particle-associated phage can be related to filter effluent
turbidity, particle counts, or measures of filter robustness;
(iv) To assess to what extent the filter breakthrough of particle-associated phage may
impact post-filter UV disinfection.
2. Materials and methods
2.1 Feed tanks
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A schematic of the experimental setup is shown in Figure 1. An online 200 L tank contained a
submersible 1/15 hp pump and was filled with Toronto (Ontario, Canada) tap water that was de-
chlorinated with 30 mg/L of sodium thiosulphate (EMD Chemicals, Gibbstown, NJ). Tap water
was chosen over distilled water due to the relatively large volumes of water required for the filter
cycle trials. The chlorine residual (free and total) was verified to be less than the detection level
of 0.01 mg/L by conducting a DPD analysis (Hach® Method 8167, equivalent to Standard
Method 4500-Cl G) with a Hach DR850® colorimeter (Hach Company, Loveland, CO). The
addition of sodium thiosulphate did not reduce the UV transmittance (UVT) significantly – the
de-chlorinated tap water had > 90% UVT in all cases.
Two interchangeable online tanks were used so that one tank fed the filter while the particle
and phage conditions were being prepared in the other tank. When the water level in the online
tank neared the bottom the pump was shutoff and quickly transferred to the second tank. During
the transfer, the filter column was fed from the head of water (30 cm) above the anthracite. In
this way the filter cycle proceeded uninterrupted. No sudden spikes in turbidity or particle counts
were observed due to the transfer from one tank to the other (e.g. see filter effluent turbidity
trends in Figure 2).
Water was pumped from the inlet tank to the top of the filter column with the water then
flowing onto the filter column by gravity, as in previous filtration studies that examined
pathogen removal by granular media filters (Huck et al., 2001; Harrington et al., 2003;
Xagoraraki et al., 2004). An overflow line positioned 30 cm above the top of the anthracite layer
allowed a constant head above the filter to be maintained. The overflow water was re-circulated
back to the inlet tank (see Figure 1). As such, the filter was operated as a declining rate filter.
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The filtration rate typically started at a rate of 700 mL/min (6.8 m/h) but decreased to 200
mL/min (1.9 m/h) by the end of the filter cycle as the filter became clogged with particles. The
filtration rate was monitored using a flow meter and control valve on the effluent line. It should
be noted that alternate filtration methods, such as using higher loading rates (e.g. > 10 m/h) or
constant rate filtration instead of declining rate filtration, may exert greater stresses on floc
particles than the declining filtration applied in this study.
Based on observations from previous studies, it was believed that pumping might break up
floc particles (Harrington et al., 2003). However this could not be confirmed via particle size or
count data, due to the small size of the particles (1-2 µm) even before pumping and due to the
size measurement threshold of the particle counter that was used (described further below).
Turbidity of the water before and after pumping/re-circulation was compared and was not
different to a statistically significant degree (α = 0.05). Further, samples taken from the influent
tank were compared with samples from the re-circulation line (i.e. after being pumped out of the
tank) in terms of percent phage particle-associated. The values were statistically similar before
and after pumping, suggesting that the pumping of water from the influent tank did not change
the percent of phage that were particle-associated.
2.2 Filter column characteristics
The filter column specifications are summarized in Table 1. Typical values for media sizes,
depths, and uniformity coefficients were selected (Castro et al., 2005). The filter media depths
(anthracite/sand depths of 45/30 cm) were similar to those used in Huck et al. (2001) (45/25 cm
at bench-scale), Harrington et al. (2003) (46/31 cm), and Becker et al. (2004) (50/25 cm), and to
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those listed as typical for dual-media filters in Kawamura (1999) (50/25 cm). The filter column
diameter (89 mm) was chosen such that there was an 89:1 ratio of column diameter to filter
media diameter (i.e. effective size of anthracite was 1.0 mm), which is greater than the minimum
50:1 ratio recommended by Rao et al. (1988) and Lang et al. (1993). The filter column diameter
was comparable in size to that used in the recent pathogen removal study by Xagoraraki et al.
(2004) (80 mm). The peak filtration rate at the beginning of each cycle (6.8 m/h) represents the
typical upper range for rapid-sand filtration (Castro et al., 2005) and is within the range of
filtration rates considered in recent studies of pathogen removal by dual-media filtration (Emelko
et al., 2001; Huck et al., 2001; Harrington et al., 2003).
Unused anthracite (Anthrafilter Filter Media, Niagara Falls, NY) and silica sand (Best Sand,
Chardon, OH) was obtained from personnel at the Ajax Water Treatment Plant (Ajax, ON,
Canada). New filter media was used in each trial to avoid microbial and particle cross-
contamination between trials. The media was thoroughly washed with at least five bed volumes
of distilled water to remove fines before each trial. Sufficient removal of fines was checked by
iteratively comparing the turbidity of the filter effluent distilled water to the turbidity of the
influent distilled water until they were approximately the same. The filter media effective sizes
and uniformity coefficients were obtained from the Ajax plant operating personnel (Table 1).
2.3. Phage growth and enumeration techniques
The methods used to grow and enumerate bacteriophages MS2 and T4 and their host
organisms have been described elsewhere (Templeton et al., 2005) and followed those described
in USEPA (2001).
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2.4 Preparation and seeding of particle-associated phage
In each trial, the particle type was either kaolin clay (Mallinckrodt Baker Inc., Phillipsburg,
NJ) (inorganic) or Aldrich® humic acid (Aldrich Chemical Company, Milwaukee, WI) (organic)
and the phage was either bacteriophage MS2 or T4. Three replicate filter cycles were conducted
for each phage-particle combination, for a total of 12 filter cycles.
The particle and phage were mixed together in two liters of distilled water contained in a
square jar beaker using a standard Phipps and Bird (Richmond, VA) jar test apparatus, as in
Templeton et al. (2005). The purpose of this step was to encourage phage particle-association
and create a concentrated solution of particle-associated phage that could then be added to the
200 L influent tank. Either 0.7 g of Aldrich® humic acid or 0.75 g of kaolin clay was added to
the distilled water. These particles were selected to represent organic and inorganic particles
present in source waters as in previous studies, and to be consistent with an earlier study
conducted by the authors (Templeton et al., 2005). While the particles used in this study are
intended as representations of natural particles, different particle attachment and phage survival
mechanisms may occur in natural waters. For example, the phage in this study were initially
free-floating and were forced to become attached to particles, whereas phage may enter the
natural environment already deeply occluded within particles, such as from fecal material.
Particles were first added to the distilled water and mixed at 100 RPM for three minutes using
a paddle mixer. Next, the phage was added and mixed at 100 RPM for three minutes followed by
20 minutes of mixing at 30 RPM. The spiked phage concentration in the two-liter jar was
approximately 106-107 PFU/mL (i.e. 10 mL of a 1010 PFU/mL phage stock solution was added to
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jar). Phage particle-association was therefore encouraged by physically bringing the phage and
particles into contact with each and allowing time for them to interact. When the water level in
the online influent feed tank neared the top of the submersible pump, the jar procedure was
started again to prepare particle-associated phage for feeding into the offline tank and
coagulation/flocculation in the offline tank, so that the filter cycle continued uninterrupted.
At the end of the jar test step, the contents of the two-liter jar were poured slowly and evenly
across the surface of the water in the 200 L tank that served as the first online influent tank and
allowed to mix. This resulted in a starting phage concentration in the 200 L inlet tank of
approximately 104-105 PFU/mL. Once the particle-associated phage was added to the influent
tank, alum was dosed at 25 mg/L for the kaolin trials and 50 mg/L for the humic acid trials.
These alum doses were determined from earlier jar experiments to be the optimal doses for
achieving the lowest subsequent settled water turbidity (i.e. best floc formation) for each water
matrix. The pH of the water (7.2 ± 0.1) was the same in all trials and no pH adjustment was
required to optimize coagulation. The pH did not change with the addition of coagulant. The
water was then mixed at 100 RPM for one minute and 30 RPM for 20 minutes using a variable
speed motor with flocculator paddles. This coagulation/flocculation regime is similar to that used
in previous studies (Krasner and Amy, 1995; Nasser et al., 1995). The resulting turbidity in the
200 L influent tank (after floc formation) ranged from 4.4 to 9.2 NTU, with a mean among all
trials of 6.4 ± 1.5 NTU.
2.5 Quantifying phage particle-association
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An elution method was used to release phages from particles for enumeration. The method
involved addition of a 2% (w/v) beef extract solution (pH 9.0) and blending at 20,000 rpm for
three minutes, similar to previously described methods (Wellings et al., 1976; Parker and Darby,
1995; Chauret et al., 1999). A comparison of parallel eluted and non-eluted samples was used to
quantify the percentage of total phage population that was particle-associated, as explained
elsewhere (Templeton et al., 2005). Initial spike and recovery experiments using both phages
showed that this method yielded high phage recoveries (> 95%) and did not affect the viability of
the phage. The blending did not alter the temperature of the samples by more than 2 ºC in any
trial. The same elution method was used for both bacteriophages MS2 and T4.
Three types of quality assurance and control tests were conducted on the blending method
beforehand to ensure that the method did not affect the viability of the phage and that the method
successfully recovered a large percentage of the particle-associated phage in each sample. These
included: (i) tests in which samples with phage but without any particles were blended to
confirm that there was no increase or decrease in phage counts (i.e. which would have indicated
either particle-association or clumping, in the case of an increase in phage counts, or phage
inactivation due to the blending method, in the case of a decrease in phage counts); (ii) tests in
which samples without any phage were blended to confirm no increase in phage counts due to
the blending method or due to cross-contamination between samples; and (iii) tests involving
blending of samples with particles and phage were present and the total known number of phage
injected in the system was known (i.e. by knowing precisely the phage concentration in the stock
solution and the precise volume added to the jars), such that it could be confirmed that all of the
particle-associated phage had been released by the elution method (i.e. by doing a ‘phage
balance’ on the system and summing the number of dispersed and particle-associated phage).
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2.6 Water quality measurements
The pH, turbidity, particle counts, and temperature of the influent water were measured over
three stages of the filter cycle: (i) during filter ripening, defined as the initial period of the filter
cycle when filter effluent turbidity exceeded the 0.3 NTU treatment goal but was generally < 1
NTU, (ii) during the stable operation period, when filter effluent turbidity was meeting the
treatment goal of < 0.3 NTU, and (iii) at the end of the filter cycle, when particle breakthrough
caused filter effluent turbidity > 0.3 NTU, but generally < 1 NTU. A filter effluent turbidity of
0.3 NTU was chosen to define the filter cycle stages since this was the level that was observed to
be consistently achieved by the bench-scale filter setup during stable operation. It should be
noted that many filter effluent turbidity regulations require < 0.1 NTU and this is achievable by
full-scale filters, however the bench-scale filter configuration could not achieve values < 0.1
NTU consistently. The filter effluent turbidity and particle counts were measured by taking grab
samples manually, with increased samples taken during periods of special interest such as during
filter ripening and end of cycle particle breakthrough. Turbidity was measured using a Hach
2100N turbidimeter (Hach Company, Loveland, CO). Particle counts were measured using a
Beckman Coulter Multisizer 3 (Beckman Coulter Canada, Mississauga, ON).
There was a concern that the particle count data might be of limited usefulness due to the
minimum particle size threshold associated with the particle counter (1 μm). Based on earlier
transmission electron microscopy (TEM) imaging (Templeton et al., 2005) and existing
knowledge of the particle size range of the particles considered in this study, most of the
colloidal kaolin and humic floc particles were expected to be smaller than this threshold.
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Turbidity measurements were therefore relied upon as the primary indication of particle
breakthrough. Headloss was also recorded by measuring the increasing water head (in
centimeters) in each of the filter media layers, as an additional indication of the progression of
the filter cycle.
Samples were also collected for phage enumeration during the three periods of interest in the
filter cycle – filter ripening, stable filter operation, and at the end of cycle breakthrough. Three
50 mL filter effluent samples were taken during each period. Parallel influent samples were
taken when filter effluent samples were collected so that instantaneous log removal of the phage
across the filter could be calculated. All samples were assessed for both dispersed and particle-
associated phage.
2.7 UV collimated beam exposures
Twenty milliliters (20 mL) of each effluent sample collected in each filter period (ripening,
stable operation, end of cycle) was exposed to UV light (254 nm) using a low pressure UV
collimated beam. The purpose of these exposures was to investigate the influence of particle-
associated phage in the filter effluent on UV disinfection. A UV dose (or ‘fluence’) of 40
mJ/cm2, typical of that applied in municipal drinking water treatment (Linden et al., 2002), was
applied to all samples. This dose was expected to result in approximately 2- to 2.5-log
inactivation of bacteriophage MS2 (NWRI, 2000; Templeton et al., 2005) and much higher
inactivation of bacteriophage T4 (> 4-log inactivation of phage T4 is achieved at only 7 mJ/cm 2
– Templeton et al., 2005). Since phage concentrations in the filter effluent were expected to be in
the range of 102-103 PFU/mL (i.e. ~3-log virus reduction across the filter) it was anticipated that
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complete inactivation of the phage might be achieved following UV exposure, especially in the
case of the UV-sensitive bacteriophage T4. As such, in cases where no viable phage remained
post-UV, the influent phage concentration was used to calculate at least the level of reduction
that was achieved by filtration and UV disinfection. For example, in cases where the influent
phage concentration was 105 PFU/mL and there were no viable phage following filtration and
UV disinfection (i.e. 0 PFU/mL), the log reduction was reported as at least 5-log reduction. This
was indicated by an arrow pointing upwards from the data points (see Figures 8 and 9).
Standard UV dose measurement and calculation methods for UV collimated beam studies, as
described by Bolton and Linden (2003), were followed. The samples were contained in 8.5
centimeter diameter Petri dishes and gently stirred using a one centimeter long stir bar during all
exposures. Samples were gently transferred to Petri dishes using wide-bore pipettes, to minimize
particle shearing (although, as described above, this could not be confirmed using the particle
analyzer equipment available). The UV intensity of the collimated beam was measured using an
IL1700 radiometer equipped with an SUD240 sensor (International Light, Newburyport, MA,
USA). A CE3055 spectrophotometer mounted with an integrating sphere sensor (Cecil
Instruments Ltd., Cambridge, UK) was used to measure the UV absorbance of the water samples.
The integrating sphere sensor captures light that is reflected off particles (light which is still able
to disinfect) and accounts for it in the UV absorbance measurement. Without this sensor, only
the light passing directly through the sample at 180º to the direction of the incident light beam is
measured. As such the UV absorbance of water samples containing particles would be
overestimated when using a spectrophotometer that is not equipped with an integrating sphere
sensor (Christensen and Linden, 2003).
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2.8 Quality assurance and quality control
New filter media was used for each trial to avoid cross-contamination of phage and particles
between trials. The interior surfaces of the walls of the empty filter column were also thoroughly
rinsed with tap water before re-filling the column with new media. Non-detect phage
enumeration samples (i.e. 0 PFU/mL) were collected from the filter effluent while running de-
chlorinated tap water (without phage) through the new media. Phage-seeded waters were also
passed through the empty filter column (i.e. no filter media) to confirm that there was no removal
of phage through the system by means other than the filter media (e.g. potentially by adsorption
to the column walls). All analytical instruments (e.g. turbidimeter, particle counter, pH meter)
were calibrated beforehand as per manufacturer specifications.
For the UV exposure component of the experiments, control exposures of bacteriophage MS2
or bacteriophage T4 in particle-free, phosphate-buffered Milli-Q® water were conducted to
confirm that both phages behaved according to their known UV dose-response relationships (i.e.
to ensure that they were not abnormally weak or UV-resistant), which are described elsewhere
(NWRI, 2000; Templeton et al., 2005).
2.9 Data presentation
‘Box and whisker’ plots are used to summarize the data for the log phage removal by filtration
and the log phage reduction by combined filtration and UV disinfection. The top of the box
represents the 75th percentile value of the data set, the bottom of the box represents the 25 th
percentile value, and the ‘whiskers’ extend to the maximum and minimum values of the data set.
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This type of plot is useful for displaying the entire spread of a data set, especially when the data
may not necessarily conform to a normal distribution.
As mentioned above, each phage-particle filter trial combination (e.g. MS2-kaolin) was
conducted three times with three samples collected at each of the periods of interest (e.g.
ripening, stable operation, end of cycle) of each filter cycle for conducting the UV exposures (i.e.
nine replicates per data point).
3. Results and Discussion
3.1 Turbidity, headloss, and particle counts
Turbidity profiles for the trials with kaolin clay are shown in Figure 2. (The turbidity profiles
for the trials with humic acid followed similar trends but are not shown here, for conciseness.)
The turbidity measurements over the replicate trials are shown together in these plots to illustrate
the reproducibility of the trends that were observed among the filter cycles. It took between 17
and 20 hours to reach the end of the filter cycle (i.e. when breakthrough caused the filter effluent
turbidity to be > 0.3 NTU) in each case. The ripening period for both types of particle was
between one and two hours long. Since the turbidity measurements were performed with grab
samples and the experiments ran unattended overnight, there was no data collected throughout
much of the stable filter operation period. The turbidity of the samples collected during the
ripening and the end of filter cycle periods were typically between 0.3 and 1 NTU.
The particle counter measurements often did not follow the turbidity measurement trends
shown in Figure 2. For example, samples taken during stable filtration period often had lower
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turbidity than samples taken during the ripening or end of cycle periods of the same trial, yet the
particle counts in the stable period samples were occasionally higher. It was not expected that
there would necessarily be a relationship between turbidity, a measure of light transmittance, and
particle concentrations. The difficulties experienced with particle counting in this study may
have been due to most of the colloidal floc particles being smaller than the particle size threshold
of the particle counting instrument (1 μm), as had been previously suggested from TEM imaging
(Templeton et al., 2005). Particle size distributions produced by the particle analyzer counted
very few particles in the > 2 µm size range, with the peak of the particle size distribution rising
steeply at the 1 µm minimum size threshold, for the unfiltered inlet samples as well as the filter
effluent samples. Most clay particles, including kaolin, are known to be <1-2 µm in size
(National Research Council, 1977; McCarthy and Zachara, 1989). The fact that no larger
particles were measured suggests that there was either poor floc formation or that the floc were
sheared during the particle count analysis. Particle counts in the filter effluent ranged from 858 to
50,943 total measurable particle counts (i.e. > 1 µm) per mL over the course of the filter cycles,
however there were no discernible pattern relating the particle counts in each filter cycle period
to the corresponding turbidity measurements. Therefore turbidity was used as the primary
indicator of the progression of the filter cycle in these trials. However, while the particle count
analysis was not successful due to the nature of the fragile, colloidal (< 1 μm) floc particles in
this particular study, it should be noted that particle counting is generally recognized as a
valuable tool for monitoring filtration performance in full-scale treatment.
The headloss within each layer increased steadily over the course of the filter cycle as the
media became clogged with particles (Fig. 3), as expected. Figure 3 shows the headloss profiles
for the six trials with AHA floc, however similar headloss values and trends were also observed
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in the trials with kaolin clay (not shown for the sake of conciseness). The sand layer exhibited
higher headloss over the course of the filter cycle than the anthracite layer due to the sand being
finer and less porous; the headloss at the end of the filter cycle was typically between 50 and 70
cm for the sand layer and 30 to 50 cm for the anthracite layer, for both particle types. This
illustrates how recording the headloss values associated with end of cycle particle breakthrough
conditions can allow a critical headloss value to be defined (on a case-specific basis) at which a
filter cycle should be stopped.
The pH (7.2 ± 0.1) and temperature (20 ± 0.1 ºC) of the influent de-chlorinated tap water was
also monitored. These parameters did not change with the addition of particles, phage, or
coagulant and remained constant over the filter column trials.
3.2 Turbidity robustness index (TRI95)
Filter performance was also assessed for robustness, as defined by the 95 th percentile turbidity
robustness index (TRI95). Robustness was calculated for the ripening, stable, and end of cycle
periods (Table 2). The T95/T50 term is an indication of variability in performance while the
T50/Tgoal term is an indicator of overall success at meeting the treatment goal. The higher the
TRI95 term the less robust the filter. During stable operation the filter performance could be
characterized as robust, with TRI95 values around 1.0. Interestingly, the least stable period (i.e.
highest T95/T50) and least robust period (i.e. highest TRI95) for almost all the filter cycles was the
ripening period rather than the end of cycle (breakthrough period), shown as gray rows in Table
2. This is significant because it will be shown that the removal of phage was approximately equal
during the ripening and end of cycle periods (i.e. not statistically different) and in some cases the
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end of cycle period had even lower phage removal than in the ripening period (Figures 6 and 7).
This suggests that while elevated TRI95 values relative to the stable operation TRI95 values may
be a rough indicator of compromised removal of particle-associated viruses during so-called ‘off-
spec’ periods of a filter cycle (when the filter effluent turbidity goal is exceeded), turbidity
robustness indices cannot serve as surrogate measurements of the degree of removal of either
dispersed or particle-associated viruses by filtration.
3.3 Bacteriophage removal by filtration
The removal of bacteriophages T4 and MS2 during the filter trials is summarized in Figures 4
and 5, respectively. Removal of bacteriophage T4 was highest during the stable operation period
and was lower during the ripening and end of cycle periods to a statistically significant degree (t-
test at 95% confidence level, α = 0.05). The same was true for the bacteriophage MS2 trials with
Aldrich® humic acid (AHA), but not for the trials with kaolin (Fig. 5). In the trials with kaolin
clay, removal of phage MS2 at the end of the filter cycle was between that observed during
ripening and also during stable operation.
Figures 6 and 7 show the percent of each phage population that was particle associated in the
influent water and in the filter effluent at the various periods of the filter cycle. Bacteriophage
MS2 became almost completely particle-associated (i.e. 99%) with both particle types in the
influent water. The percent of the bacteriophage T4 that was particle-associated in the influent
water was generally not as high as the particle-association of bacteriophage MS2 and varied
more among replicates. Further, samples taken from the influent tank over the course of each
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filter trials showed no statistically significant time-dependent variations in the percentage of the
phage population that was particle-associated.
The percent of phage MS2 that was particle-associated in the filter effluent was lower (α =
0.05) than in the influent water for both particle types during the ripening and stable operation
periods of the filter cycle, especially in the trials with Aldrich® humic acid floc (Fig. 7). This
means that more of the dispersed phage penetrated the filter relative to the particle-associated
phage in those periods. In other words, the particle-associated phage were effectively removed
by the filter media while the smaller, dispersed phage (i.e. 25 nm bacteriophage MS2 and 200
nm bacteriophage T4) passed through the media. This was also the case for bacteriophage T4
with the AHA floc particles, but not so with the kaolin clay particles for which the majority (i.e.
> 80%) of the bacteriophage T4 in the filter effluent during stable operation was still particle-
associated.
The percent of each phage that was particle-associated in the filter effluent was generally
higher at the end of the filter cycle than during the stable operation, although this could not be
supported statistically (α = 0.05) due to variability in the data among the replicates. The increase
in phage particle-association in the filter effluent during breakthrough conditions may have been
due to the release of previously accumulated particle-associated phage from the filter media.
In addition, samples were collected from ports at the bottom of each of the anthracite and sand
layers to investigate the removal of phage by each layer of media during stable filter operation.
This experiment (conducted with bacteriophage T4 and kaolin only) indicated that each of the
two media layers contributed approximately equal removal of phage (0.6 to 1.0-log removal
each) (Table 3).
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3.4 Impact of filter breakthrough on UV disinfection
Figures 8 and 9 show the combined reduction of phage by filtration and UV disinfection (40
mJ/cm2). The arrows pointing upward on the top of the whiskers indicate that these values show
at least the amount of log reduction that was achieved, as explained earlier (see Section 2.7). In
those cases, no viable phage remained following the UV disinfection step. This was expected for
bacteriophage T4, which is a very UV-sensitive phage. The authors have shown that a 4-log
inactivation of bacteriophage T4 is achieved at a UV dose of only 5 mJ/cm2 (Templeton et al.,
2005). For bacteriophage MS2, which is more UV-resistant, viable phage post-UV were counted
at the end of the filter cycles with Aldrich® humic acid (AHA). In the trials with AHA, the
overall reduction of phage MS2 was lower at the end of a filter cycle period when compared to
the ripening and stable operation stages, to a statistically significant degree (α = 0.05). This was
despite the fact that the turbidity levels in the ripening and end of cycle were approximately the
same. The negative effects on UV disinfection were observed during the humic acid trials but not
during the kaolin trials was likely due to the higher UV absorbance of the humic acid relative to
the clay particles, as reported in Templeton et al. (2005).
In addition, samples of the unfiltered influent waters with turbidities ranging from 4.4 to 9.4
NTU (mean 6.4 ± 1.5 NTU) were exposed to the UV collimated beam (Figure 10) for
comparison purposes. As mentioned above, a previously study (Templeton et al. 2005)
considered the impact of unfiltered waters on UV disinfection, however at much higher turbidity
values (up to 100 NTU), since that study aimed to examine the fundamental impacts of particle
composition (e.g. UV absorbing content) and therefore sought to generate easily measurable
impacts. In the current study, however, turbidities were more representative of what may be
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encountered in unfiltered water supplies, and so a more realistic impact on UV disinfection by
particle-associated phage could be assessed.
The UV transmittance (UVT) of the unfiltered samples with humic acid ranged from 69 to
81% and the UVT of the unfiltered samples with kaolin clay ranged from 80 to 89%. However,
the UV transmittance of the samples was taken into account in the UV dose calculation (i.e. more
exposure time was given to samples with lower UVT, in order to achieve the same applied UV
dose).
As a comparison with the UV disinfection of the filter effluent samples, unfiltered samples
(from the influent tank) were also exposed to UV disinfection. A previous study (Templeton et
al., 2005) showed that humic-containing floc particles have the potential to shield phage from
UV light at elevated turbidites (up to 100 NTU), but the turbidities of the unfiltered water in the
present study were much lower (< 10 NTU). For the unfiltered samples, post-UV viable phage
were present in countable numbers (i.e. no arrows shown pointing upwards on Figure 10 data),
since the initial pre-UV concentrations of phage were higher than in the filter effluent.
Interestingly, bacteriophage T4 survivors were counted post-UV even though this phage is very
sensitive to UV light (Templeton et al., 2005). For bacteriophage MS2, a more UV-resistant
phage, the UV inactivation in the presence of kaolin clay particles was comparable to that
achieved in particle-free water (NWRI, 2000; Templeton et al., 2005), however the inactivation
in the presence of UV-absorbing AHA floc was reduced to a statistically significant degree
(Figure 10), which agreed with previous findings at higher turbidities (Templeton et al. 2005).
Therefore, this shows that the particles that were present in the unfiltered influent water, even at
relatively low turbidities < 10 NTU, had the potential to have a measurable UV-shielding impact
on the phage should they have penetrated the filter.
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4. Conclusions
Dual-media filtration can provide an effective means of reducing particle-associated viruses in
drinking water, which may otherwise be shielded from UV light. Greater than 2.5-log removal
(99.68%) of both phages was achieved during the stable filter operation stages of all the trials
(i.e. defined as when filter effluent turbidity was < 0.3 NTU), with as high as 4-log phage
removal achieved in some trials. Increased breakthrough of particle-associated viruses occurs
during ‘off-spec’ periods of the filter cycle (when filter effluent turbidity exceeded 0.3 NTU),
such as at the end of the cycle. These periods of breakthrough of viruses and particle-associated
correspond with increased filter effluent turbidity. Elevated turbidity robustness index (TRI95)
values relative to the robustness values for stable filter operation can be used as a rough indicator
of the increased likelihood of breakthrough of particle-associated viruses during ‘off-spec’ filter
periods. However, it cannot be used as a quantitative predictor, since there were no direct link
observed between robustness and the breakthrough of particle-associated phage. UV disinfection
of the filter effluent was impeded during ‘off-spec’ filter periods, especially at the end of a filter
cycle. Utilities should therefore be careful to practice filter-to-waste during off-spec filter periods
(i.e. when filter effluent turbidity exceeds the goal) in order to prevent the breakthrough of UV-
protected particle-associated viruses. Further research is recommended to consider the filter
removal of other particle-associated microorganisms using different filtration regimes (e.g.
higher loading rates), other coagulants (e.g. ferric chloride), and different particle types and
sizes. The ability of chemical disinfectants (e.g. chlorine, chloramines, ozone) to inactivate
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particle-shielded viruses should also be considered under conditions that are representative of
treatment practice.
Acknowledgements
The authors acknowledge the funding support of that Natural Sciences and Engineering
Research Council of Canada (NSERC). The authors also thank Richard Jones of the Region of
Durham (Ajax Water Treatment Plant) for providing filter media and Sida Ren and Carole
Baxter for their assistance in the laboratory.
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